DNA: The Genetic Material PDF

Genetics– BIO310 DNA: The Genetic Material CH 5.1 Organization of Functional Sites Along Bacterial Chromosomes  Chromosomes and Genomes Chromosomes are the structures that contain the genetic material The genome comprises all the genetic material that an organism poss...

Genetics– BIO310 DNA: The Genetic Material CH 5.1 Organization of Functional Sites Along Bacterial Chromosomes  Chromosomes and Genomes Chromosomes are the structures that contain the genetic material The genome comprises all the genetic material that an organism possesses ◦ In bacteria, it is typically a single circular chromosome ◦ In eukaryotes, a nuclear genome refers to one complete set of nuclear chromosomes ◦ Note: ◦ Eukaryotes also possess a mitochondrial genome ◦ Plants also have a chloroplast genome 3 Function of Genetic Material The main function of the genetic material is to store information required to produce an organism ◦ The DNA molecule does that through its base sequence DNA sequences are necessary for ◦ Synthesis of RNA and cellular proteins ◦ Replication of chromosomes ◦ Proper segregation of chromosomes ◦ Compaction of chromosomes (so they fit in the cell) 4  Bacterial Chromosomes Usually, a circular molecule that is a few million bps long ◦ Escherichia coli, ~ 4.6 million base pairs ◦ Haemophilus influenzae, ~ 1.8 million base pairs A typical bacterial chromosome contains a few thousand different genes ◦ Protein-encoding genes (structural genes) account for the majority of bacterial DNA The nontranscribed DNA segments between genes are called intergenic regions ◦ Repetitive sequences may play roles in DNA folding, gene regulation, and genetic recombination Origin of replication – initiation site for DNA replication 5 6  Structure of Bacterial Chromosomes The bacterial chromosome is found in a region of the cell called the nucleoid The nucleoid is not surrounded by a membrane ◦ So, the DNA is in direct contact with the cytoplasm 10  Compaction To fit within the bacterial cell, the chromosomal DNA must be compacted about a 1000-fold Bacterial chromosome has a central core with loops called microdomains emanating from the core Typically 10,000 bp in length; an E. coli chromosome has 400 to 500 microdomains Adjacent microdomains organized into macrodomains 800 to 1000 kbp in length Nucleoid-associated proteins (NAPs) form the micro and macrodomains Bend DNA or act as bridges between DNA regions  DNA Supercoiling Twisting forces change conformation of DNA Two strands of DNA coil around each other, and the formation of additional coils due to twisting forces is called DNA supercoiling Both underwinding and overwinding of the DNA double helix can induce supercoiling DNA structures that differ in supercoiling are called topoisomers of each other Source: Adapted from Wang, Xindan, Llopis, Paula Montero, and Rudner, David Z. (2013), Organization and Segregation of Bacterial Chromosomes, Nature Reviews Genetics, vol. 14, no. 3, 191–203. 12 Bing Videos Bing Videos Bing Videos DNA Supercoiling Affects Chromosome Function The chromosomal DNA in Negative supercoiling bacteria is negatively supercoiled In E. coli, there is one negative supercoil per 40 Helps in the compaction of the chromosome turns of the double helix Creates tension that may be released by DNA strand separation This enhances DNA replication and transcription 16 Control of Supercoiling Supercoiling in bacteria is controlled primarily by ◦ DNA gyrase (topoisomerase II) ◦ Creates negative supercoils using energy from ATP ◦ Can also relax positive supercoils when they occur ◦ DNA topoisomerase I ◦ Relaxes negative supercoils ◦ Breaks one strand and rotates the DNA The competing action of these two enzymes governs the overall supercoiling of bacterial DNA  Supercoiling Enzymes as Drug Targets The ability of gyrase to introduce negative supercoils into DNA is crucial for bacteria to survive ◦ So this enzyme can be targeted to cure bacterial diseases Two main classes of drugs inhibit bacterial topoisomerases, but do not inhibit eukaryotic ones ◦ Quinolones ◦ Coumarins An example of a quinolone is ciprofloxacin (“Cipro”) ◦ Used in the treatment of anthrax, among others Organization of Functional Sites Along Eukaryotic Chromosomes  Eukaryotic C hromosomes Eukaryotic species contain one or more sets of chromosomes Each set is composed of several different linear chromosomes Chromosomes in eukaryotes are located in the nucleus Eukaryotic chromosomes are tens of millions to hundreds of millions of bp in length  Organization of Eukaryotic Chromosomes A eukaryotic chromosome contains a long, linear DNA molecule Origins of replication – many per chromosome Centromere – constricted region of the chromosome, which has a role in chromosome segregation during mitosis and meiosis Kinetochore proteins – group of proteins that link centromere to spindle apparatus Telomere – at ends, prevent translocations and are necessary for maintenance of chromosome length 22 Eukaryotic Genes Genes are located between the centromeric and telomeric regions along the entire chromosome A single chromosome usually has a few hundred to several thousand genes In less complex eukaryotes (such as yeast) Genes are relatively small They contain primarily the sequences encoding the polypeptides That is, Very few introns are present In more complex eukaryotes (such as mammals) Genes are long They tend to have many introns – noncoding intervening sequences Intron lengths range from less than 100 to more than 10,000 bp Sizes of Eukaryotic Genomes and Repetitive Sequences Variation in size of eukaryotic genomes Repetitive sequence and how they affect genome sizes  Sizes of Eukaryotic Genomes Genome sizes vary greatly between species – sometimes because of more genes BUT… Variation is not necessarily related to complexity ◦ Example: Salamander species ◦ There is a two-fold difference in the size of the genome in two closely related salamander species ◦ Size variation is not because of extra genes - It is due to accumulation of repetitive DNA seqs ◦ Seqs that do not encode proteins (b) ©Ann & Rob Simpson; (c) ©Gary Nafis Has a genome that is more than twice as large as that of P. richmondi 26  Sequence complexity refers to the number of times a particular base Sequence sequence appears in the genome C omplexity There are three main types of DNA sequences Unique or non- Moderately Highly repetitive repetitive repetitive 28 Unique and Repetitive Sequences Unique or non-repetitive sequences Found once or a few times in the genome Includes protein coding genes as well as introns and other noncoding DNA In humans, make up roughly 41% of the genome Moderately repetitive Found a few hundred to a few thousand times Includes Genes for rRNA Transposable elements – short segments of DNA that can move within the genome  Unique and Repetitive Sequences Highly repetitive ◦ Found tens of thousands to millions of times ◦ Some are transposable elements ◦ Example: Alu family in humans ◦ Others are clustered together in tandem arrays ◦ Example: AATAT and AATATAT in Drosophila ◦ Commonly found in the centromeric regions ◦ Function is not understood; sequences and amount of repetitive DNA can vary even among closely related species Transposition The organization of How transposons and The effects of sequences within retrotransposons transposable different types of move to new elements on gene transposable locations in a function elements genome  Transposition Transposition is a process in which a DNA segment is inserted into a new location ◦Can occur at many different locations within the genome. ◦The DNA segments are transposable elements (TEs) ◦ Sometimes referred to as “jumping genes” TEs were first identified by Barbara McClintock in the early 1950s from her classical studies in corn ◦ Since then, many different types of TEs have been found in species as diverse as bacteria, fungi, plants and animals  Transposition Pathways Two basic transposition pathways have been identified ◦ Simple transposition ◦ The TE is removed from original site and transferred to a new site by a “cut and paste” mechanism ◦ These TEs are called transposons ◦ Retrotransposition ◦ The TE moves via retrotransposition – TE is transcribed into RNA, then reverse transcriptase makes a second copy in DNA ◦ These TEs are called retrotransposons or retroelements ◦ Increase in number during retrotransposition 34 Access the text alternative for slide images. 35.  Simple Transposons Simple Transposons In addition to Flanking direct repeats Inverted repeats Transposase gene The simple transposon also carries one or more genes not necessary for transposition Example: Antibiotic resistance gene 38 Retrotransposons LTR Retrotransposons Related to retroviruses Move around the genome, but cannot produce viral particles Contain long terminal repeats (LTRs) at both ends A few hundred bp long Encode reverse transcriptase and integrase Non-LTR Retrotransposons Less like retroviruses May encode reverse transcriptase / endonuclease Some derived from normal eukaryotic genes Ex: Alu in humans derived from 7SL RNA gene 40  Autonomous versus Non autonomous Elements TEs are complete (or autonomous) when they contain all the information necessary for transposition to occur TEs are incomplete (or nonautonomous) when they lack a gene that is necessary for transposition to occur Example: In corn Ac or Activator element is autonomous Ds element is nonautonomous - it lacks transposase and needs transposase from Ac element to move  Transposase The enzyme transposase catalyzes the removal of a TE and the its reinsertion at another location: Transposase monomers bind IR sequences at the end of the TE Monomers dimerize, bringing IRs together DNA is cleaved between the IRs and DRs, which excises TE from the chromosome Transposase carries TE to a new site and cleaves the target DNA at staggered recognition sites; TE inserted and ligated to target DNA 48 Transposons | Transposable elements | Types of transposons| how transposons work?  They are in the same direction and are repeated at both ends of the element 50  Increasing the number of TEs Even simple transposition can increase the number of transposons in the genome Transposition often occurs around time of replication ◦ One of these TEs can transpose ahead of the fork where it is copied again. ◦ One genome will still have one TE, but the other will now have two copies 46  Reverse Transcriptase Retroelements use an RNA intermediate in their transposition mechanism The movement of retroelements also requires two key enzymes in the following steps: ◦ Retrotransposon is transcribed into RNA ◦ Reverse transcriptase uses RNA as a template to make dsDNA ◦ LTRs at the end of the dsDNA are recognized by integrase which cuts the target site and catalyzes the insertion of the TE into this site 48  Transposable Elements Influence on Mutation and Evolution Over the past few decades, researchers have found that transposable elements probably occur in the genomes of all species Can rapidly enter genome and proliferate Many different kinds  TABLE 12.1 Examples of Transposable Elements Element Type Approximate Description Length (bp) Bacteria IS1 Transposon 768 An insertion element that is commonly found in five to eight copies in E. coli Tn10 Transposon 9300 One of many different bacterial transposons that carries antibiotic resistance Yeast Ty elements Retrotransposon 6300 Found in S. cerevisiae in about 35 copies per genome Drosophila P elements Transposon 500–3000 A transposon that may be found in 30–50 copies in P strains of Drosophila. It is absent from M strains. Humans Alu Retrotransposon 300 A retrotransposon found in about 1 million copies in the human sequence genome L1 Retrotransposon 6500 A retrotransposon found in about 500,000 copies in the human genome Plants Ac/Ds Transposon 4500 Ac is an autonomous transposon found in corn and other plant species. It carries a transposase-encoding gene. Ds is a nonautonomous version that lacks a functional transposase- encoding gene. 56  TABLE 12.2 Abundance of Transposable Elements in the Genomes of Selected Species Percentage of the Total Genome Species Composed of TEs* Frog (Xenopus laevis) 77 Corn (Zea mays) 60 Human (Homo sapiens) 45 Mouse (Mus musculus) 40 Fruit fly (Drosophila melanogaster) 20 Nematode (Caenorhabditis elegans) 12 Yeast (Saccharomyces cerevisiae) 4 Bacterium (Escherichia coli) 0.3 *In some cases, the abundance of TEs may vary somewhat among different strains of the same species. The values reported here are typical values. 57 Biological Significance of TEs The biological significance of transposons in evolution remains a matter of debate ◦ Selfish DNA hypothesis ◦ TEs exist because they can ◦ Like parasites, can proliferate in host as long as they do not overly harm the host ◦ TEs offer an advantage ◦ Transpositions events are deleterious so TEs they would be eliminated from genome if they didn’t over an advantage ◦ In bacteria, may carry antibiotic-resistance genes ◦ May cause insertion of exons into the coding region of other genes, providing new functions – exon shuffling 58 Negative Effects of TEs Transposons have a variety of effects on chromosome structure and gene expression ◦ Many outcomes are likely to be harmful; transposition can be stimulated by radiation, mutagens, hormones ◦ When transposon activity is not regulated and kept under control, they can cause chromosomal abnormalities and sterility ◦ Example: Drosophila crosses that introduce P elements to a strain with no P elements ◦ Known as hybrid dysgenesis 59  TABLE 12.3 Possible Consequences of Transposition Consequence Cause Chromosome Structure Chromosome breakage Excision of a TE Chromosomal rearrangements Homologous recombination between TEs located at different positions in the genome Gene Expression Mutation Incorrect excision of TEs Gene inactivation Insertion of a TE into a gene Alteration in gene regulation Transposition of a gene next to regulatory sequences or the transposition of regulatory sequences next to a gene Alteration in the exon content of a gene Insertion of exons into the coding sequence of a gene via TEs, a phenomenon called exon shuffling Gene duplications Insertion of a gene into a transposon that transposes to another site in the genome 54